The incidence of disease caused by pathogenic and opportunistic fungi has been increasing over the past decade. See, e.g., McNeil, M. M, et al., Clin. Infect. Dis. 33:641-47 (2001); Ampel, N. M., et al., Clin. Infect. Dis. 27:1528-30 (1998); National Nosocomial Infections Surveillance (NNIS) System report, data summary from Jan. 1990 to May 1999, Am. J. Infect. Control 27:520-32 (1999). In humans, these fungal infections are especially prevalent in people with suppressed immune systems, such as HIV-positive and severely ill patients. For example, Penicillium marneffei is the third most common cause of opportunistic infections in patients with AIDS in Thailand. Vanittanakom N., Sirisanthana T., Curr. Top. Med. Mycol. 8:3542 (1997). Additionally, P. chrysogenum and P. citrinum have been recognized as the cause of human disease.
Diagnosis of fungal infections is typically made by isolating the infective organism in culture, by serologic assays, or through histopathologic examination of tissue. See, e.g., Hamilton, A. J., Med. Mycol. 36:351-64 (1998). However, pathogenic fungi may require several weeks to grow, and a positive culture may represent benign colonization, rather than true invasion and infection, especially when opportunistic organisms are isolated. Serologic tests on a single serum sample to detect circulating antifungal antibodies may be inconclusive (especially in immunosuppressed subjects). The acquisition of paired acute and convalescent sera, which is necessary for a definitive serologic diagnosis, requires an additional 3 to 4 weeks before convalescent serum can be obtained. Morrison C. J., and Lindsley, M. D., in Fungal Pathogenesis: Principles and Clinical Applications (New York: Marcel Dekker Inc., 2001; Calderone R. A., and Cihlar R. L., eds.). Therefore, histopathologic examination of tissue sections was often the most rapid, and sometimes the only, method available to diagnose invasive fungal disease. However, histopathologic diagnosis of fungal infections is usually made through morphologic criteria, and fungi with atypical morphologic features can be difficult to identify and diagnose. In addition, fungi for which different anti-fungal therapies could be used often look morphologically similar in tissue sections.
The relatively recent development of automated DNA synthesis has allowed production of molecular probes with consistently defined properties that may result in increased test sensitivity, specificity, and reproducibility. Past research in the molecular identification of fungi has typically concentrated on a single species or genus of a fungus. See, e.g., LoBuglio, K. F., and J. W. Taylor, J. Clin. Microbiol. 33:85-89 (1995); Loffler, J., et al., Med. Mycol. 36:275-79 (1998). For example, U.S. Pat. Nos. 5,631,132; 5,426,027; 5,635,353; and 5,645,992; and PCT publication WO 98/50584, disclose nucleic acid probes and methods for detecting fungal species based on a certain region (the ITS2 region) of rDNA. Additionally, some methods of molecular identification of fungi can be very difficult or cumbersome to perform, or require expensive, specialized equipment. See, e.g., Sandhu, G. S., et al., J. Clin. Microbiol. 35:1894-96 (1997); Sandhu, G. S., et al., J. Clin. Microbiol. 33:2913-19 (1995); and Turenne, C. Y., et al., J. Clin. Microbiol. 37:1846-51 (1999).